In this work, a reusable DNA sensing microchip was developed for detection of vomitoxin (deoxynivalenol, DON) in sorghum using Cd-based core-shell CdSe@CdS quantum dots (QDs) as promising electrochemiluminescence (ECL) emitter. The size-adjustable aqueous phase CdSe@CdS QDs were prepared through homogeneous method, exhibiting strong cathodic ECL emission with a central wavelength of 520 nm in S2O82− coreactant. And gold nanoparticles-modified iron cobalt cyanide hydrate (Fe–Co–Au) was introduced as an accelerator to amplify the ECL signal. ECL signal was quenched after the formation of a double-stranded (dsDNA) S1–S2 by generating an electron transfer system between the emitter and ferrocene (Fc), which are modified on the aptamer (ssDNA S1) and its complement sequence (ssDNA S2), respectively. When the target DON is presence, the aptamer ssDNA S1 will bind to the DON and trigger the unbinding of double strands DNA and the release of the ssDNA S2, thus the signal can be generated. This approach offers a feasible method for the detection of DON within the range of 1 ng/mL to 200 ng/mL.